An Unexpected Encounter: Respiratory Syncytial Virus Nonstructural Protein 1 Interacts with Mediator Subunit MED25

ABSTRACT Human respiratory syncytial virus (RSV) is the leading cause of severe acute lower respiratory tract infections in infants worldwide. Nonstructural protein NS1 of RSV modulates the host innate immune response by acting as an antagonist of type I and type III interferon (IFN) production and signaling in multiple ways. Likely, NS1 performs this function by interacting with different host proteins. In order to obtain a comprehensive overview of the NS1 interaction partners, we performed three complementary protein-protein interaction screens, i.e., BioID, MAPPIT, and KISS. To closely mimic a natural infection, the BioID proximity screen was performed using a recombinant RSV in which the NS1 protein is fused to a biotin ligase. Remarkably, MED25, a subunit of the Mediator complex, was identified in all three performed screening methods as a potential NS1-interacting protein. We confirmed the interaction between MED25 and RSV NS1 by coimmunoprecipitation, not only upon overexpression of NS1 but also with endogenous NS1 during RSV infection. We also demonstrate that the replication of RSV can be enhanced in MED25 knockout A549 cells, suggesting a potential antiviral role of MED25 during RSV infection. Mediator subunits function as transcriptional coactivators and are involved in transcriptional regulation of their target genes. Therefore, the interaction between RSV NS1 and cellular MED25 might be beneficial for RSV during infection by affecting host transcription and the host immune response to infection. IMPORTANCE Innate immune responses, including the production of type I and III interferons, play a crucial role in the first line of defense against RSV infection. However, only a poor induction of type I IFNs is observed during RSV infection, suggesting that RSV has evolved mechanisms to prevent type I IFN expression by the infected host cell. A unique RSV protein, NS1, is largely responsible for this effect, probably through interaction with multiple host proteins. A better understanding of the interactions that occur between RSV NS1 and host proteins may help to identify targets for an effective antiviral therapy. We addressed this question by performing three complementary protein-protein interaction screens and identified MED25 as an RSV NS1-interacting protein. We propose a role in innate anti-RSV defense for this Mediator complex subunit.

. NS1 proxeome hits identified by BioID. Columns from left to right show the 139 UniProt accession number, the gene name, the log2 (RSV NS1-BirA*-Flag RSV/ RSV mKate2-BirA*-Flag 140 RSV) fold change value and -log p value as depicted in Fig. 2B  (hits_Q_below0.35) shows a subset of tab 1 where only preys are retained that give a Q value below 158 0.35. The third tab (filtered_list) shows a subset of tab 2, where only preys are retained with particle 159 counts higher than two and where known aspecific binders are removed. 160 In the ORF prey collection, several empty preys are present. These preys have not been sequenced and 161 are therefore considered as empty. When we upload the filtered datasets (q value <0.35, particle 162 counts > 2, removal of known aspecific binders) of the MAPPIT and KISS primary screen into the jvenn 163 online tool to identify common proteins between the performed screens, all empty preys are 164 considered as one prey since no distinction is made by the tool. This accounts for the small differences 165 in protein numbers that are listed in this filtered list (388 preys in Suppl.

Q9UI47 CTNNA3
May be involved in formation of stretch-resistant cell-cell adhesion complexes.

P36897 TGFBR1
Transmembrane serine/threonine kinase forming with the TGFbeta type II serine/threonine kinase receptor.

Q6IPU0 CENPP
Component of the CENPA-CAD (nucleosome distal) complex, a complex recruited to centromeres which is involved in assembly of kinetochore proteins, mitotic progression and chromosome segregation.

Q5T5P2 KIAA1217
Required for normal development of intervertebral disks.

Q9NR12 PDLIM7
May function as a scaffold on which the coordinated assembly of proteins can occur.

Q9H4G0 EPB41L1
Involved in actin binding and actomyosin structure organization.

P40937 RFC5
Smallest subunit of the replication factor C complex, which is required for DNA replication.

Q5MJ09 SPANXN3
Belongs to the SPAN-X family.

Q86TB9 PATL1
RNA-binding protein involved in deadenylation-dependent decapping of mRNAs, leading to the degradation of mRNAs.

P61201 COPS2
Essential component of the COP9 signalosome complex (CSN), an essential regulator of the ubiquitin (Ubl) conjugation pathway.

P01350 GAST
Gastrin stimulates the stomach mucosa to produce and secrete hydrochloric acid and the pancreas to secrete its digestive enzymes.

Q96JC1 VPS39
Plays a role in vesicle-mediated protein trafficking to lysosomal compartments including the endocytic membrane transport and autophagic pathways.

Q86W26 NLRP10
Involved in the innate immune response by contributing to proinflammatory cytokine release in response to invasive bacterial infection.

Q96PU4 UHRF2
E3 ubiquitin ligase that plays important roles in DNA methylation, histone modifications, cell cycle and DNA repair.

P42357 HAL
Involved in the histidine catabolic process.

Q8NCS7 SLC44A5
Involved in choline transmembrane transporter activity.